Differential effects of iron load on basal and interferon-gamma plus lipopolysaccharide enhance anticryptococcal activity by the murine microglial cell line BV-2.

Here we evaluated the influence of intracellular iron levels on the constitutive and interferon (IFN)-gamma plus lipopolysaccharide (LPS) enhanced anticryptococcal activity by the murine microglial cell line BV-2. We demonstrated that iron loading via ferric nitrilotriacetate (FeNTA) resulted in a significant increase in the constitutive levels of anticryptococcal activity, while the enhancing effects by IFN-gamma plus LPS were prevented. Accordingly, a major increase was observed in the levels of thiobarbituric reactive substance (TBARS) produced upon iron loading under basal conditions, whereas IFN-gamma plus LPS treatment, that per se did not affect TBARS production, prevented by about 50% the enhancement otherwise occurring in response to iron loading. The potential involvement of multiple effector system and their relation to intracellular iron will be discussed.

Enhanced resistance to Cryptococcus neoformans infection induced by chloroquine in a murine model of meningoencephalitis.

Although the pathogenesis of cerebral cryptococcosis is poorly understood, local immune cells, such as microglia and astrocytes, likely play a critical role in containing infection. Chloroquine (CQ) is a weak base that accumulates within acidic vacuoles and increases their pH. Consequently, proteolytic activity of lysosomal enzymes and intracellular iron release/availability are impaired, resulting in decreased availability of nutrients crucial to microorganism survival and growth in the host. We found that CQ enhances BV2 microglial-cell-mediated anticryptococcal activity in vitro. The phenomenon is (i) evident when both unopsonized and opsonized microorganisms are used and (ii) mimicked by NH4Cl, another weak base, and by bafilomycin A1, an inhibitor of vacuolar-type H+-ATPases. In vivo, intracerebral administration of CQ before lethal local challenge with Cryptococcus neoformans results in a significant augmentation of median survival time and a marked reduction of yeast growth in the brain and is associated with the enhancement of local interleukin 1beta (IL-1beta) and IL-6 mRNA transcripts. Overall, these results provide the first evidence that CQ enhances anticryptococcal host defenses.

Iron regulates microglial cell-mediated secretory and effector functions.

Iron homeostasis and macrophage physiology are tightly intertwined. In the present study, we evaluated the influence of iron loading on the constitutive and interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-induced functional and secretory properties of microglial cells, using the in vitro established murine cell line BV-2. We demonstrate that iron augments the basal and IFN-gamma plus LPS-enhanced anti-Candida albicans activity exerted by BV-2 cells and that the phenomenon occurs with no enhancement of phagocytic activity. Furthermore, when the secretory properties of IFN-gamma plus LPS-treated BV-2 cells were assessed, we found that tumor necrosis factor remains unchanged while nitric oxide production is significantly reduced in iron-loaded cells. The addition of the iron chelator deferiprone (L1) reverts the effects of iron on BV-2 functional and secretory properties. These data suggest that iron differently affects secretory and effector functions of BV-2 microglial cells, thus implying that iron interferes with murine microglial cell physiology.

Biomolecular events involved in the establishment of brain anticandidal resistance.

Using a murine model, we have demonstrated the establishment of cerebral resistance to local lethal challenge with Candida albicans strain CA-6, by previous intracerebral (i.c.) infection with the low-virulent strain PCA-2. Here we show that i.c. infection with PCA-2 is effective in drastically reducing brain colonization following secondary infection with CA-6. As assessed by colony forming unit assay and histopathological analysis, microbial counts are impaired, granuloma formation and hyphal growth are also reduced in brains of PCA-2- and CA-6-infected mice with respect to CA-6-challenged mice. Furthermore, using PCR studies, we found that, while PCA-2 (i.e. healing infection) induces transient cytokine gene expression in the mouse brain, CA-6 lethal challenge results in long-lasting (until mouse death) high levels of all cytokine gene transcripts assessed. Finally brains from mice that will resist CA-6 challenge, because of previous infection with PCA-2, also exhibit a transient induction of all cytokine genes. Only IL-1 beta remains highly expressed at all time- points tested. Overall, these results provide evidence that healing and non-healing C. albicans i.c. infections differ in the immune reaction(s) locally evoked, at least in terms of cytokine gene expression, strongly suggesting cytokine involvement in the establishment of brain anticandidal resistance.

Role of nitric oxide and melanogenesis in the accomplishment of anticryptococcal activity by the BV-2 microglial cell line.

In the present paper, we investigated the involvement of cryptococcal melanogenesis and macrophage nitric oxide (NO) production in the accomplishment of anticryptococcal activity by microglial effector cells, using the murine cell line BV-2. We demonstrate that the constitutive levels of anticryptococcal activity exerted by BV-2 cells is significantly enhanced upon interferon gamma plus lipopolysaccharide treatment. The phenomenon, which occurs with no enhancement of phagocytic activity, is associated with the production of high levels of NO and is abolished by addition of NG-monomethyl-L-arginine. Comparable patterns of results are observed employing either unopsonized or opsonized microbial targets, the latter microorganisms being markedly more susceptible to BV-2 cell antimicrobial activity. Furthermore, melanization of Cryptococcus neoformans significantly reduces its susceptibility to BV-2 antimicrobial activity, regardless of the fact that activated macrophages or opsonized microorganisms have been employed. In conclusion, our results provide evidence that NO-dependent events are involved in the fulfillment of anticryptococcal activity by activated microglial cells and that fungal melanization is a precious escamotage through which C. neoformans overcomes host defenses.

Biomolecular events involved in anticryptococcal resistance in the brain.

We have recently shown that intracerebral (i.c.) administration of heat-killed Cryptococcus neoformans (HCN) enhances mouse resistance to a subsequent local challenge with lethal doses of viable yeast cells. Here we show that i.c. administration of HCN is also effective in significantly delaying brain colonization of mice intravenously infected with viable C. neoformans. PCR analysis revealed that interleukin 6 (IL-6) and IL-1 beta gene expression occurs in brain of HCN-treated mice but not in brains of saline-treated controls. In contrast, no differences are observed in terms of tumor necrosis factor alpha and IL-1 alpha gene transcripts, which are slightly and highly detectable, respectively, in saline-treated mice and which remain such also following HCN treatment. Furthermore, i.c. administration of exogenous IL-6 or IL-1 beta, but not tumor necrosis factor alpha, before local challenge with viable C. neoformans results in significantly reduced microbial counts in the brain and blood and in increased mouse survival. Taken together, these observations provide initial evidence that brain anticryptococcal resistance involves elicitation of a local cytokine response, involving primarily IL-6 and IL-1 beta.

Different events involved in the induction of macrophage tumor necrosis factor by Candida albicans and lipopolysaccharide.

Using an in vitro experimental model, we have recently demonstrated that Candida albicans in its hyphal form (H-Candida), similarly to lipopolysaccharide (LPS), enhances tumor necrosis factor (TNF) secretory response in the cloned macrophage (M phi) population ANA-1. Here we show that H-Candida and LPS each differ in their requirements for intact protein kinase functions, susceptibility to 0.4-microns micropore-size membranes, and sensitivity to polymyxin B. These results, together with the synergistic effect occurring between H-Candida and LPS in inducing TNF response, indicate the existence of different receptor(s) and/or signal-transduction pathway(s) through which the two stimuli act.